Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Predominance of phage group II in Staphylococcus strains isolated from humans

2545 strains of Staphylococcus aureus isolated from 24 Polish laboratories in the years 1994-95 were investigated. Phage typing was performed according to the method of Blair and Williams using the present basic set of typing phages and additional phages 88, 89 and 187. The phages were employed in concentrations of RTD and 100 x RTD. The predominance of phage group II, reported elsewhere since 1980-ties, was found in the present study (23.6%). Strains of group III were second in frequency (16.6%), whereas strains of groups I and V, as well as type 95 occurred in small percentage (7.6%, 4% and 3.4% respectively). Strains of groups II and V have been rarely lysed by phages belonging to other groups. The use of additional phages resulted in typability of strains by 7.9%. Percentage of non-typable strains was high and amounted to 22.0%.     

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.     

Bacteriophage typing of Listeria species.

A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment.     

Phage types of Staphylococcus aureus strains and their antibiotic resistance in carriers of medical students population

The phage types of 78 S. aureus strains isolated from nose swabs obtained from a medical students in 2005 -2006 was determined and antibiotic resistance of the phage types was analysed. 680 students were tested in order to obtain the strains and 11.5% of them were carriers of S. aureus. Phage typing was performed using basic set of23 phages and 3 additional phages: 88, 89 and 187. Drug resistance was determined by the disc-diffusion method. The most frequent in studied population were the group III (21.8%) and strains lysed by phages belonging to varied groups (21.8%). Highly different phage patterns were observed among strains belonging to each of the group. Strains belonging to the group III as the strains lysed by phages from varied groups were most frequently resistant only to penicillin (52,9% respectively). Resistance to penicillin was also most often observed in the strains belonging to another groups and phage types. Usefulness of the additional phages 88,89 and 187 was in the investigations as no more than 51% of strains was lysed by this phages.     

A new scheme for phage typing Salmonella bareilly and characterization of typing phages

A new phage typing scheme using wild bacteriophages isolated from sewage for phage typing Salmonella bareilly is described. Six hundred and thirty-seven strains of Salm. bareilly could be separated into 11 different phage types using five wild phages. Overall typability was 94.5%. These phages belonged to two different morphotypes. A1 and B1, and showed varying host range.     

A new scheme for phage typing Salmonella bareilly and characterization of typing phages

A new phage typing scheme using wild bacteriophages isolated from sewage for phage typing Salmonella bareilly is described. Six hundred and thirty-seven strains of Salm. bareilly could be separated into 11 different phage types using five wild phages. Overall typability was 94˙5%. These phages belonged to two different morphotypes, A1 and B1, and showed varying host range.     

Fecal carriage of Escherichia coli O157:H7 and carcass contamination in cattle at slaughter in northern Italy.

Feedlot cattle slaughtered at a large abattoir in northern Italy during 2002 were examined for intestinal carriage and carcass contamination with Escherichia coli O157:H7. Carcass samples were taken following the excision method described in the Decision 471/2001/EC, and fecal material was taken from the colon of the calves after evisceration. Bacteria were isolated and identified according to the MFLP-80 and MFLP-90 procedures (Food Directorate's Health Canada's). Eighty-eight non-sorbitol-fermenting E. coli O157:H7 isolates were obtained from 12 of the 45 calves examined. In particular, E. coli O157:H7 isolates were found in 11 (24%) fecal and five (11%) carcass samples. PCR analysis showed that all 11 fecal samples and five carcass samples carried eae-gamma1-positive E. coli O157:H7 isolates. In addition, genes encoding Shigatoxins were detected in O157:H7 isolates from nine and two of those 11 fecal and five carcasses, respectively. A representative group of 32 E. coli O157:H7 isolates was analyzed by phage typing and DNA macrorestriction fragment analysis (PFGE). Five phage types (PT8, PT32v, PT32, PT54, and PT not typable) and seven (I-VII) distinct restriction patterns of similarity >85% were detected. Up to three different O157:H7 strains in an individual fecal sample and up to four from the same animal could be isolated. These findings provide evidence of the epidemiological importance of subtyping more than one isolate from the same sample. Phage typing together with PFGE proved to be very useful tools to detect cross-contamination among carcasses and should therefore be included in HACCP programs at abattoirs. The results showed that the same PFGE-phage type E. coli O157:H7 profile was detected in the fecal and carcass samples from an animal, and also in two more carcasses corresponding to two animals slaughtered the same day.     

Use of WARD et al's scheme for bacteriophage typing of Salmonella enteritidis strains isolated in Poland

The typing phages set of Ward et al. was used to type a total of 517 strains of Salmonella Enteritidis isolated in Poland in 1986-1995. According to the Ward et al. scheme, 56.5% of the strains tested were assigned to 14 different phage types. Phage types 8, 4, 1 and 4a were placed first, second, third and fourth, respectively. They were dominated both in the outbreak isolates and in the isolates from the other sources. Ten phage types were represented by single strains. Other strains reacted with phages without showing any of the designated phage type (37.1%) or were untypable (6.4%). The Ward et al. scheme seems to demonstrate not enough high degree of strain discrimination for Salmonella Enteritidis isolates in Poland. It seems that the Ward et al. scheme is not enough useful for the epidemiological investigations of Salmonella Enteritidis in Poland.     

Comparative characteristics of antigenic conversion induced by Shigella flexneri and Escherichia coli 0129 phages

The interaction of S. flexneri converting phages PE5, P90 and fV with E. coli antigenic variant O129, E. coli O129 converting phage VB with the above antigenic variant and with S. flexneri y-variant was studied. Phage PE5 and phage VB were found to induce the conversion of O-antigen in E. coli antigenic variant 0129 and in S. flexneri y-variant with the detection of antigens V and 7,8. Phages P90 and fV induced no conversion of O-antigen. Changes in the antigenic properties of convertants were confirmed by the results obtained in the agglutination test and in the agglutination adsorption test.     

Evaluation of the usefulness of new international experimental phages for typing methicillin resistant Staphylococcus aureus (MRSA)

The aim of the study was to determine the usefulness of the set of experimental phages obtained from the Central Public Health Laboratory in London for typing of MRSA strains in Poland. The study was performed on 150 MRSA strains isolated from various clinical materials in various regions of the country. The set of 10 experimental phages and the international basic set of 23 phages were used for typing. The results of the study showed that 76.8% of MRSA strains were typing with the experimental set of phages. The frequency of inhibition reactions was 19.9%. Only 3.3% of the strains were nontypable with the new phages while nearly half of the studied strains were nontypable with the basic set of phages. The studied strains were divided into 19 phagotypes. There was a high frequency of typable strains among MRSA typable and nontypable strains and those inhibited by the basic set of phages (71.4%-85.7%). These data indicate that the set of 10 experimental phages is useful for typing of MRSA strains isolated in Poland except for phage M3 which failed to react with almost all the strains and should be excluded from the proposed set.     

Relationship between capsular types 5 and 8 and phage types in Staphylococcus aureus isolates from cow, goat and ewe milk

Abstract In order to check the relationship between capsular polysaccharide (CP) type 5 and 8 and certain phage patterns, previously described in human Staphylococcus aureus clinical isolates, we typed 100 CP types 5 and 8 S. aureus strains isolated from cow, goat and ewe milk, with the human set of phages. The proportion of typable strains was much less than that found with human strains. The association between CP types 5 and 8 and phage patterns reported for human isolates was only partially confirmed and an original correlation between susceptibility to group III phages and CP type 5 was found.     

Further Studies on Staphylococci in Meats, : IV. The Bacteriophage Pattern and Antibiotic Sensitivity of Isolates from Nonfrozen Meats

Of 272 isolates of Staphylococcus aureus recovered from 173 samples of 10 market meats from 27 stores, 173 (63.2%) were phage typable, employing 28 phages. Sixty per cent of the phage-typable strains belonged to group III, followed by 14.5% to mixed groups I and III, 10.4% to Group I, 8.7% to all mixed groups, 4.6% to group II, and 1.7% to group IV. The most commonly recovered patterns were 83, 53/83, and other similar combinations of 53. The nonpigmented strains which did not have bound coagulase were less phage sensitive than the pigmented strains having bound coagulase. None of the isolates were resistant to novobiocin, kanamycin, chloramphenicol, and erythromycin. Twenty-three per cent were resistant to streptomycin, 17% to ristocetin, 11% to penicillin, and 4.4% to chlortetracycline. The phage types are compared to those of other food and human isolates and found not to differ too greatly. Their possible origins into the meats are discussed.     

Phage typing ofstaphylococcus aureus strains using experimentalS. aureus phage 292

The characteristics of a newStaphylococcus aureus phage, 292, are described. Phage 292 appears closely related to typing phage 96 of the basic international series and is serologically related to the group B staphylococcal phages. In typing studies, phage 292 further subdivided 44.4% (120/270) of the type 94/96 strains and 42.0% (68/162) of the type 96 strains. A comparison of the typing efficacy of phage 292 and four other phages (14, 15, 16, 17) is also presented.     

Evaluation of the usefulness of three collections of bacteriophages for typing methicillin-resistant Staphylococcus aureus,isolated in Moscow hospitals

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.     

Phage types and plasmid profiles of plasmid DNA strains of Salmonella enterica subsp. enterica ser. Enteritidis (S. Enteritidis) isolated from food poisoning outbreaks in 2001

Salmonella Enteritidis strains are the most often isolated Salmonella serovar in Poland. In the present study, phage typing, antibiotic resistance testing and plasmid profile analysis, have been applied to characterise 41 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 41 S. Enteritidis strains coming from Poland. All 41 strains were typable and 5 different phage types were observed. Among 41 strains tested, both PT6 and PT21 were recognized in the 15 strains (36.6%). Nine strains (22%) belonged to phage type 8. The others PTs were represented by small amount of strains (PT1var and PT4). Among all tested isolates only 4 different plasmid profiles were observed. Of the 41 strains investigated, 16 (39%) contained the 57 kb plasmid alone. The remaining 25 strains (61%) except 57 kb plasmid, possessed additional DNA particles. The probable phage type conversion of PT21 to PT1var strain, possibly connected with smaller DNA particle presence, was observed. This hypothesis needs confirmation. The real S. Enteritidis epidemiological situation in Poland should be known after introducing of systematic, annual research program.     

Analysis of whole genome sequencing for the Escherichia coli O157:H7 typing phages

Background

Shiga toxin producing Escherichia coli O157 can cause severe bloody diarrhea and haemolytic uraemic syndrome. Phage typing of E. coli O157 facilitates public health surveillance and outbreak investigations, certain phage types are more likely to occupy specific niches and are associated with specific age groups and disease severity. The aim of this study was to analyse the genome sequences of 16 (fourteen T4 and two T7) E. coli O157 typing phages and to determine the genes responsible for the subtle differences in phage type profiles.

Results

The typing phages were sequenced using paired-end Illumina sequencing at The Genome Analysis Centre and the Animal Health and Veterinary Laboratories Agency and bioinformatics programs including Velvet, Brig and Easyfig were used to analyse them. A two-way Euclidian cluster analysis highlighted the associations between groups of phage types and typing phages. The analysis showed that the T7 typing phages (9 and 10) differed by only three genes and that the T4 typing phages formed three distinct groups of similar genomic sequences: Group 1 (1, 8, 11, 12 and 15, 16), Group 2 (3, 6, 7 and 13) and Group 3 (2, 4, 5 and 14). The E. coli O157 phage typing scheme exhibited a significantly modular network linked to the genetic similarity of each group showing that these groups are specialised to infect a subset of phage types.

Conclusion

Sequencing the typing phage has enabled us to identify the variable genes within each group and to determine how this corresponds to changes in phage type.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1470-z) contains supplementary material, which is available to authorized users.     

Staphylococcal and other Bacterial Species Associated with Intramammary Infections in Danish Dairy Herds

Four thousand six hundred forty– five quarter milk samples from 1179 cows from 20 commercial dairy herds were examined in order to determine the prevalence of bacterial species. A total of 859 isolates from 839 (18.1%) culture positive samples could be assigned to 34 different species and subspecies. Diagnostics of staphylococcal species was based on conventional procedures able to differentiate between all 36 species and subspecies presently acknowledged. Staphylococcus aureus was found in 10.2% of the samples and was the most common species isolated. Streptococcus dysgalactiae (1.6%) and Streptococcus uberis (1.4%) were the second and third most common species isolated. Seventeen different coagulase negative staphylococcal species (CNS) were found in 4.1% of the samples. The most frequently isolated CNS were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%). Isolates of S. aureus were phage typed, and isolates of S. epidermidis were investigated by phage typing, antibiogram typing, and biotyping. A total of 378 (79.9%) isolates of S. aureus could be typed by phages, assigning them to 18 different phage types. However, 6 phage types accounted for 92.1% of the typable isolates. One to 2 phage types predominated within each herd. Eleven (18%) isolates of S. epidermidis could be typed by phages, assigning the isolates to 3 different types. Biotyping of S. epidermidis produced a total of 8 different types, the most common accounting for 29.5% of the isolates. A total of 6 different antibiogram types were observed among all isolates of S. epidermidis. Resistance towards penicillin (36.1%), tetracycline (9.8%) and streptomycin (9.8%), were recorded in the isolates of S. epidermidis. However, 35 (57.4%) of the isolates were susceptible to all 12 antibiotics tested.     

Salmonella serotype typhimurium phage typing. A critical evaluation

Of 12,930 Salmonella serotype typhimurium strains, phage typed during 1985-1988, 45.68% were "nontypable" by Anderson's set; the percent of typable strains decreased from 54.17 in 1986 to 30.54 in 1988. Of 90 phage patterns of sensitivity, 22 were currently encountered. Phage types 1, 18 and 104 were most frequent to strain of both human and non-human origin. In food generating S. typhimurium outbreaks, phage types 1 and 36 were prevalent. Except lysotypes 198 and 95, isolated from "single cases" in man only, all other phage types were common in man and animals, too. Introducing other typing methods to serotype typhimurium "nontypable" strains (by Anderson's set) was considered necessary for epidemiological purposes.     

Phage types, plasmid profiles and chromosomal restriction profiles of Salmonella enterica subsp. enterica ser. enteritidis (S. enteritidis) isolated in Poland in 1999-2000]

Salmonella Enteritidis strains are the most often isolated Salmonella serovars in Poland. In the present study, phage typing, plasmid profile analysis, and PFGE have been applied to characterize 140 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 140 S. Enteritidis strains coming from Poland. All 140 strains were typable and six different phage types were observed. A total of 125 (89%) of 140 isolates examined belonged to PT 4. The others PTs were represented by small amount of strains (PT1-2, PT6-6, PT7-1, PT8-4 and PT21-2 strains). Among all tested isolates six different plasmid profiles were observed. Of the 140 examined strains, 128 (91.4%) contained the 57 kb plasmid alone. After XbaI digestion four distinct pulse field chromosomal restriction profiles among studied S. Enteritidis were observed. XbaI and SpeI chromosomal restriction profiles of S. Enteritidis PT4 were identical with reference strain profiles. Our findings confirmed earlier suggestions that the increase of human salmonellosis cases in Poland was caused by S. Enteritidis PT4 and was due to consumption of contaminated food. This study confirmed the importance of using PFGE in combination with phage typing, plasmid typing and antibiotic resistance testing for studying the epidemiology of S. Enteritidis.